CHAPTER ONE

1.0.0 INTRODUCTIONThe rapid growth in industrialization in the last century has caused a dramatic increase in energy consumption all over the world and so the world is in need of alternative sources of energy to meet industrial and transportation needs. Crude oil is known to account for over 50% of the energy needs in the world today however it is a limited source of energy (Yang and Wyman 2008, Sun and Cheng 2002).

Bioethanol is a promising alternative source of energy for the limited crude oil. It is believed to be the most important renewable fuel in terms of volume and market value (Licht 2006). While the production of ethanol from Starch and sugar based raw materials such as cereals, tubers, sugarcane and sugar beets is not new however in recent times the production of ethanol from lignocellulosic biomasss waste has acquired a significant interest. The process of ethanol production from these sources is summarized in the figure 1.1 below.

Figure 1.1 Raw materials for ethanol production (Gunasekaran and Chandrarag,1999); Ethanol production from plant biomass and agroby-products may involve other possible routes like the simultaneous saccharification and fermentation where the hydrolysis and fermentation reactions are carried out in the presence of lignin.

Reports have shown that the United States of America and Brazil are the top producers of ethanol from corn and sugarcane respectively. Both countries

account for the production of 89% of the world’s production in 2009 (www.ethanolrfa.org/page/objects/pdf/RFAoutlook2010 retrieved 2010-04-17). The use of starch and sugar based materials brings an added challenge considering the rapid rise in world population as these materials also serve as sources for food. This has forced the current interest in the use of lignocellulose biomass as raw materials for bioethanol production.

Lignocellulose is believed to be the most abundant plant material resource with an estimated annual production of 1 x 1010 metric tonnes and these materials form a large bulk of biomass. There are loosely classified as Agricultural residues, forest/wood wastes, municipal wastes (papers) and energy crops dedicated to ethanol and other biofuel production (Sanchez and Cardona 2008).

The presence of lignin, hemicelluloses and cellulose in most lignocellulose materials makes access to these polymers by enzymes very difficult and the challenge for scientist over the past three decades has been to access these polymers and convert them to fuels. Considering the recalcitrant and heterogenous nature of lignocellulosic biomass, it is generally agreed that the following processes for conversion of lignocelluloses biomass is required; size reduction, pretreatment and enzymatic hydrolysis by cellulases and other hydrolytic enzymes. The sugars generated from the hydrolysis are then fermented by microorganisms (yeast or other ethanologenic organisms) into ethanol.

Although extensive research has been carried out on pretreatment, enzymatic hydrolysis and fermentation of biomass material, very few studies exist on materials that contain starch in addition to cellulose, hemicelluloses and lignin. A few of such materials include sorghum bran, potatoe peels, yam peels, cassava peels and a host of other agricultural residues.

This research will focus on the enzymatic digestion of cassava peels for bioethanol production. This is because it is becoming increasingly difficult to ignore the enormous waste generated from cassava processing in the tropics. Cassava(Manihot esculata) is a woody shrub extensively cultivated as an annual crop in tropical regions of the world for its edible starchy root. It is known as mandioca (brazil), Yuca (Columbia), kamoleng kahoy(philipines) mushu(china) akpu, ege or ugburu (Nigeria) and man sampalang (Thailand). Cassava peels is a

byproduct from the processing of cassava either for human consumption or starch production. The peel of the cassava is 1-4mm thick and account for 10-14% of the total dry matter of the root. (Adegbola et al 1996, Nartey 1979). Food and Agricultural organization (FAO) reports show that Nigeria alone produces 38 million tonnes of cassava per annum as at 2004 and this would in turn generate about 4 million tonnes of cassava waste per annum. These figures are increasing yearly with a plan to produce bioethanol from cassava tubers. While a small amount of the waste which include the peels, leaves and unused leftover stalks from the processing of cassava is used as animal feed and also as manure in small farms in the rural area, much of the waste is burnt or thrown away. The need to convert this waste into biofuel becomes imperative.

Several studies on pretreatment and enzymatic hydrolysis of several biomass waste (wheat straw, corn Stover, rice straw) have been carried out ( Sun and Cheng 2002, Taherzadeh and Kirimi 2008, Yang and Wyman 2008). It is generally agreed that substrate properties like cellulose fibre crystallinity, porosity(accessible surface area), degree of polymerisation, degree of acetylation, presence of lignin amongs other factors all affect hydrolysis rate and yield. There is no existing literature on how these properties are related to hydrolysis rate in starch rich lignocelluloses waste.

This research will be the first to seek a mechanistic approach to study the kinetics of enzymatic hydrolysis of starch rich lignicellulose waste using cassava peels. Much of the research up till now on starch rich lignocelluloses biomass (corridor et al 2007, Yoonan and Kongkiattikajorn 2004, sornvoraweat and knongkiattijorn 2009) have all been empirical studies showing hydrolysis yield without any reference to the substrate properties.

Previous studies on the fermentation of this waste have all being carried out by yeast however this study will compare the fermentation capabilities of yeast and zymomonas mobilis (a gram negative bacteria) believed to have superior abilities for fermentation.

One of the main objectives of this research is to have a better understanding of the interactions between alpha amylase and cellulase enzymes and perhaps seek a clearer picture of the mechanism of the hydrolysis step so as to develop an efficient digestion model for cassava peels and similar waste.

A more detailed analysis of literature on pretreatment and enzymatic hydrolysis of lignocelluloses will be discussed in chapter two. This report will also critically look at different substrate properties that are believed to affect hydrolysis yield.

Chapter two will also look at literature on starch rich lignocellulose waste (cassava peels and sorghum bran) while chapter three will highlight the aims, objectives and scope of the research.

CHAPTER TWO

2.1.0 OVERVIEW OF LITERATURE

A large and growing body of literature abound on various aspects of lignocellulose biomass to ethanol conversion. Several notable reviews on pretreatment (Sun and Cheng 2002, Taherzadeh and Karimi 2008, Yang and Wyman 2008, Alvira et al 2009) and enzymatic hydrolysis (Boomarius et al 2008, Zhang and Lynd 2004, Mosier et al 1999, Zheng et al 2008). These reviews show that the key to efficient utilization of lignocelluloses biomass lies in the pretreatment and enzymatic hydrolysis steps.

This chapter will look at the typical structure of a lignocelluloses material, it will then review the different pretreatment methods applied to lignocellulose biomass. It will also examine the enzymatic hydrolysis of lignocelluloses materials especially on the kinetics of cellulase enzymes. It will then go on to highlight literature on starch digestion. An overview of available

literature on cassava peels hydrolysis and fermentation will be discussed.

2.2.0 STRUCTURE OF LIGNOCELLULOSE

Lignocellulose biomass is primarily composed of cellulose, hemicellulose and lignin. Cellulose is a polysaccharide consisting of a linear chain of several hundred to over a thousand β-(1-4) linked D-glucose units. Hemicellulose can be any of several heteropolymers such as arabinoxylans. It may contain many different sugar monomers like xylose, mannose, galactose, rhamnose and arabinose. Hemicellulose contains most of the D pentose sugars. Xylose is always the sugar monomer present in the largest amount in hemicelluloses when compared to the other sugars like mannose and galactose usually present in varying amounts. Hemicellulose consist of shorter chains of 500-3000 sugar units when compared to cellulose which has about 7000-15000 glucose residues per polymer. It is also a branched polymer in contrast to cellulose which is unbranched. Lignin, the third component of most lignocellulosic biomass is a complex chemical compound and the most difficult to degrade. ‘’It is an integral part of the secondary walls of plants’’ (lebo et al 2001). It is heterogenous and lacks a defined primary structure. It fills the spaces in the cell walls of plants between cellulose, hemicellulose and pectin components. Unlike cellulose and hemicellulose it is hydrophobic and a non carbohydrate polymer with about 40 aromatic subunits which are covalently linked(Wardrop 1969, McClements 2005). The barrier to hydrolysis of lignocelluloses is that the sugars are trapped inside the lignocelluloses due to the crosslinking between the polysaccharides (cellulose and hemicelluloses) and the

lignin via ester and ether linkages. Ester linkages arise between oxidized sugars, the uronic acids and the phenols and propanols of the lignin. To extract the fermentable sugars one must first disconnect the cellulose from the lignin before the hydrolysis of the sugars to simple monosaccharides.

Figure 2-1. Basic structure of lignocellulose (http://www.lbl.gov/Publications/YOS/Feb/)

2.3.0 PRETREATMENT

Pretreatment is believed to be the most important unit operation in terms of cost followed closely by the cost of enzymatic hydrolysis of pretreated cellulose (Wooley et al 1999, Hyman 2007, Yang and Wyman 2008). Economic analysis show that almost 40% of projected cost is associated with releasing sugars from hemicelluloses and cellulose with pretreatment responsible for about half of this total. Studies from several reviews show that pretreatment methods can be broadly classified into three main types; physical, chemical and biological pretreatment methods. A combination of physical and chemical pretreatments gives another pretreatment method termed physico-chemical pretreatment. 2.3.1 PHYSICAL PRETREATMENT

This requires the use of mechanical comminution to reduce size by a combination of chipping, grinding and milling to reduce cellulose crystallinity or the use of pyrolysis to produce simple sugars (Sun and Cheng 2002). Several studies on irradiation by microwaves, ultrasound, electron beam and gamma rays are ongoing. However, the major drawback to the use of physical pretreatment methods is that there are believed to be far more expensive than the other pretreatment methods especially when the effect on the lignocelluloses substrate is considered (millet et al 1976).

2.3.2 BIOLOGICAL PRETREATMENT

This involves the use of microorganisms such as white, brown and soft fungi to degrade lignin and hemicelluloses materials. Although investigations have been carried out on several white rot fungi or similar microorganisms that are believed to attack mainly lignin and hemicelluloses (hataka 1983, fanet al 1987). The hydrolysis rate is very slow inspite of its advantages which include low energy requirement and favourable environmental conditions.

2.3.3 CHEMICAL PRETREATMENT

This remains the most investigated pretreatment method available to date. It includes Acid and alkaline hydrolysis, ozonolysis, organosolv( use of organic solvents; ethanol, acetone, methanol etc), oxidative delignification(peroxidise enzyme in the presence of hydrogen peroxide), use of ionic liquids, wet oxidation ( use of oxygen at high temperature) amongst others.

The use of concentrated and dilute acids have been investigated and found to be successful for the pretreatment of several biomass materials.(wheat straw, rice husk etc). Dilute acids are however preferred to concentrated acids because it

generates lower sugar degradation compounds like furfural, 5-hydroxy methyl furfural(HMF) and aromatic lignin degradation compounds. These are known to affect hydrolysis (Saha 2005,Wyman 1996).The main limitations to the use of acids is the requirement for reactors that are resistant to corrosion to be used. The difficulty in recovery of the acids used also make the process economically not feasible and this is also a challenge (silver and Zacchi 1995). Acid pretreatment is believed to solubilise a high percentage of hemicelluloses and degrade a good percentage of lignin.

Alkaline pretreatment methods using sodium, potassium and calcium hydroxides have been studied with sodium hydroxide being the most investigated. Alkaline pretreatment is believed to cause an increase in the internal surface area, a decrease in crystallinity and degree of polymerisation. A separation of structural linkages between lignin and the carbohydrate is also expected with alkaline pretreatment. Alkaline are however expensive although it has been suggested that calcium hydroxide be used instead as lime is cheap and can be recovered easily. Another advantage with alkaline pretreatment is that the reactions can be carried out at low temperatures and pressure unlike other methods ( Carvalheiro et al 2008, Mosier et al 2005, Kim and holtzapple 2006, Fan et al 1987).

Other pretreatment methods based on the use of chemicals all possess their unique advantages and shortcomings. Most studies are however inconclusive. Ozonolysis is believed to effectively remove lignin and prevents the formation of toxic residues for the subsequent hydrolysis step however it is very expensive due to the large amount of ozone required (Vidal and Molinter 1998). Organosolv is also highly favoured when the recovery of lignin is of interest (Zhao et al 2009). Several lignocelluloses materials have been treated with organic solvents (acetone, ethanol, methanol, ethylene glycol) and sometimes a mixture of these chemicals and acid catalyst. The acid catalyst is believed to accelerate the breaking of hemicellolose bonds. These solvents are however expensive and require appropriate extraction and separation techniques which lead to an added cost. These solvents also need to be removed as they inhibit hydrolysis enzymes(Sun and Cheng 2002).

Another interesting chemical pretreatment that has received considerable attention of recent is ionic liquids pretreatement. Ionic liquids are salts, they contain large organic cations and small inorganic anions which exist as liquids at

low temperature(1butyl-3-methylidazolium cation ). They are sometimes called ‘green’ liquids because no toxic gases are formed when they are used. (Alvira et al 2010). Although the use of ionic liquids have been mainly limited to pure crystalline cellulose(Yang and Wyman 2008), reports of their uses on lignocelluloses biomass such as straw(li et al 2009) and wood (lee et al 2009) exist. The low volatility and high thermal stability of these liquids makes research into their use a worthy option. Perhaps the most serious disadvantage to this pretreatment method is that for now, the process cost is not known and it is still unclear if these liquids will affect hydrolytic enzymes.

Ammonia recycle percolation(use of ammonia through a reactor packed with biomass at elevated temperature of 80-1000C), wet oxidation (oxygen at high temperature and pressure) and other oxidizing agents are continually being researched.

2.3.4 PHYSICO-CHEMICAL PRETREATMENT

These methods employ the use of chemicals in a high pressure system and a sudden explosive decompression in the vessel to bring about degradation of the cellulose, hemicelluloses and lignin transformation. This include steam explosion pretreatment, ammonia fibre explosion and CO2 explosion. Liquid hot water pretreatment is another hydrothermal treatment which does not make use of rapid decompression and does not employ any catalyst or chemicals. Pressure is applied to maintain water at elevated temperature (160-2400C) and provoke changes in the arrangement of the lignocellulose (Alvira et al 2010).

Steam explosion is believed to be the most effective pretreatment process for the pretreatment of agricultural residues and hardwood as it has a low energy requirement. In steam explosion, high pressure saturated steam is used to treat the chipped biomass at elevated temperatures of 160-2600C corresponding to pressure of 0.69-4.83MPa for several seconds to a few minutes before the material is exposed to atmospheric pressure. The major disadvantage associated with steam explosion is that it can generate compounds that may be inhibitory to enzymes and microorganisms in the downstream process. This may include weak acids like acetic acids formed from acetic groups present in the hemicellulose fraction. Partial degradation of hemicelluloses may also yield 5 hydroxymethylfurfural (HMF). Further degradation of furfural and 5

hydroxymethyl furfural also occur leading to the formation of formic and levulinic acids(palmqvist and Hahn-hagerdal 2000). Although the toxic compounds generated depends on the raw materials, it has been suggested that pretreated biomass needs to be washed with water to remove inhibitory materials however this reduces the overall saccharification yield due to the removal of sugars generated by the hydrolysis of hemicelluloses. An alternative would be to research into obtaining more tolerant microorganisms for fermentation (liu et al 2005).

Studies have shown that residence time, temperature, chip size and moisture content are the most important factors for steam explosion ( Duffa and Murray 2006).

CO2 and Ammonia Fibre Explosion (AFEX) also employ the same physical mechanism of decompression at high pressure as steam explosion. The difference being in the use of CO2 and liquid ammonia in the CO2 and Ammonia fibre explosion respectively. Both processes are not as efficient as steam explosion although they are known to produce less inhibitory compounds when compared to steam explosion (Dale et al 1984,Mes-hartree et al 1988)

Addition of sulphuric acid, SO2 or CO2 is also believed to improve the hydrolysis, decrease the production of inhibitory compounds and lead to more complete removal of hemicelluloses (Morjanoff and Gray 1987)

2.4.0 EFFECT OF PRETREATMENT METHOD ON SUBSTRATE PROPERTIES

The constituent physico-chemical properties of lignocellulosic biomaterials will determine to a large extent the type of pretreatment most appropriate to employ. A combination of two or more pretreatment methods may be appropriate so as to maximise the Saccharification process without degrading the carbohydrates.( Galbe and Zacchi 2007).

The different pretreatment methods are believed to have different sugar release patterns. Alvira et al 2008 compared the most significant effects the different pretreatment technologies have on the structure of lignocelluloses in the table below.

Milling

Steam Expolsion

LHW Acid Alkaline Oxidative AFEX Lime CO2

explosionARP

Increases accessible surface area

H H H H H H H H H H

Cellulose decrystallization

H - N.D - - N.D H N.D N.D H

Hemicelluloses solubilisation

- H H H L - M M H M

Generation of toxic compounds

- H L H L L M M - H

Table 2.1:H: high effect; M: moderate effect; L: low effect; N.D: not determined;LHW: liquid hot water AFEX:alkaline fibre explosion; ARP: ammonia recycle percolation (Alvira et al 2010)

One question that arises from these studies is which of these factors; accessible surface area, cellulose decrystallization, hemicelluloses solubilisation, lignin structure alteration or generation of toxic materials has the most effect on hydrolysis rate and yield. Although it is agreed that some of these properties are related (removal of lignin or hemicelluloses solubilisation would lead to an increase in surface area however a more systematic study that recognises the peculiar properties of a particular biomaterial and perhaps identify how the substrate properties affects hydrolysis would be of much benefit. This might perhaps help in identifying the appropriate pretreatment that will bring about the most desired change identified for the substrate.

The pretreatment methods applied to starch rich lignocelluloses biomass include milling on cassava peels (Yoonan and Kongkiattikajorn, 2004), acid and alkaline pretreatment(Sornvoraweat and Kongkiattijorn 2009). Liquid hot water has also been tested on sorghum bran (Corridor et al 2007). These researchers did not treat pretreatment in detail. Their studies was mainly focused on enzymatic hydrolysis and fermentation. Sornvoraweat and Kongkiattijorn 2006 studied the simultaneous saccharification and fermentation of cassava peels. Milling and acid/alkaline pretreatment were combined. The study is rather vague on the effect of pretreatment on hydrolysis. Moreover combining milling and acid/alkaline pretreatment would make the process expensive.

The effect of liquid hot water on sorghum (Corridor et al 2007) was believed to be profound. Starch was first degraded by alpha amylase and beta glucosidase then the substrate was pretreated with liquid hot water and then hydrolysed by a cocktail of enzymes. Results have shown that a combination of starch degradation, optimum hot water pretreatment and enzymatic hydrolysis resulted

in maximum total sugar yield of 75%. The process suggested might be cumbersome with the expected washing out of the starch after degradation, then liquid hot water being applied to the substrate before enzymatic hydrolysis is carried out. Perhaps a better experimental design would have been to treat with liquid hot water and then combine enzymatic hydrolysis with starch degradation.

The studies would also have been more complete if the effect of pretreatment on substrate properties is investigated so as to ascertain which factors have the greatest effect on enzymatic hydrolysis. The effects of pretreatment methods on starch degradation could also be investigated.

2.5.0 ENZYMATIC HYDROLYSIS OF LIGNOCELLULOSE

Extensive research has been carried out on the cellulose hydrolysis by cellulase enzymes however as stated earlier very few studies exist on combining starch and cellulose hydrolysis. Starch hydrolysis carried out by alpha amylase and glusosidase enzymes can be modelled with the michaelis menten kinetic model. The michaelis menten model which is obeyed by most enzymes assumes a homogenous system where mass transfer (substrate to enzyme and product from enzyme) is not rate limiting therefore only the catalytic step(enzyme substrate complex to enzyme product) is governing the rate of reaction. However cellulose hydrolysis occurs in a heterogeneous system of two phases; the enzymes in an acqueous phase while the cellulose exist in crystalline solid. The physical characteristics of the crystalline cellulose also complicate modelling. The accessible surface area, degree of polymerisation, degree of crystallinity, substrate and enzyme concentration all contribute significantly to the reaction rate modelling.

Researchers have shown an increased interest in the use of a cocktail of enzymes (cellulases,pectinases,xylanases,lacasses, amylase etc) to degrade lignocellulose materials. The existing accounts on starch and cellulose hydrolysis report the combined use of cellulase, alpha amylase, xylanases and pectinase for hydrolysis however the studies fail to show how these enzymes interact or specifically how alpha amylase and cellulase enzymes affect the sugar release patterns. The studies also do not take into account the effect of these enzymes on the substrate properties (degree of polymerisation, crystallinity, concentration of chain ends). A more systematic study that identifies how alpha amylase, cellulases and other

hydrolytic enzymes interact with these substrate properties would give a mechanistic approach that will help in understanding the kinetics of this reaction.

The studies would have been even more interesting if the researchers had shown how much glucose is produced from either the starch or cellulose component of the biomaterial.

2.5.1 CELLULOSE HYDROLYSIS: LITERATURE REVIEW

The enzymatic hydrolysis of cellulose by cellulase involves three types of cellulases; endoglucanases (EC.3.2.14) that randomly cleaves β -1,4-glycosidic bonds on cellulose chains away from the chain ends, Cellobiohydrolases(EC.3.2.91) produces cellobiose by attacking cellulose from chain ends. (cellobiohydrolase I acts from the reducing end while cellobiohydrolase II acts from the non reducing ends of the cellulose chain). Beta glucosidase is the third enzyme in the cellulase system which converts cellobiose to glucose. (Henrisat 1994, Lynd et al 2002, Zhang and Lynd 2004).

The complexity of this system involving three different enzymes acting on cellulose; a heterogenous solid substrate makes the kinetics of this reaction difficult to comprehend. The mechanism of this reaction is still not fully understood. Studies on the kinetics of this reaction must take into account the molecular architecture of the cellulase enzyme, the role of the substrate properties (crystallinity,degree of polymerisation,accessible area) and the decline in reaction rate that characterise this reaction(Zhang and Lynd, 2004).

Several empirical studies(Sattler et al 1989, Kim and Holltzapple 2000, Berlin et al 2007) that help in quantifying the effects of an individual substrate property and initial rate estimations are available but these do not provide much understanding on the mechanistic details of the process. Recently, researchers have shown an increased interest in mechanistic or semi-mechanistic kinetic models that take into account the adsorption of enzymes. Zhang and lynd 2004, points out the imperative need for researchers to seek a functional approach to modelling of enzymatic hydrolysis of cellulose towards identifying rate limiting factors and a deeper understanding at the level of substrate features and multiple enzyme activities.

One of the major challenges of mechanistic models have been the need for improved methodologies for determination of substrate properties like degree of polymerisation, crystallinity and cellulase accessibility. This is a particular problem with materials that have both lignin and cellulose as most measurements of these properties in literature have been on pure cellulose substrate.

Cellulase enzymes are modular proteins with two distinct independent domains; the catalytic module and the cellulose binding module(CBM). The catalytic module is responsible for the hydrolysis of the cellulose chain while the cellulose binding module has the ability to increase the adsorption of cellulolytic enzymes onto the insoluble cellulose(Mosier et al 1999). The two modules are joined by a flexible-glycosylated linker region approximately 3—44 amino acids in length and rich in pyroline, glycine, serine and threonine. Endoglucanase and cellobiohydrolases are tadpole shaped with the catalytic core forming the head and a wedge-shaped cellulose binding module at the tip of the tail.

The exact mechanism of cellulose hydrolysis is not yet known due to insufficient experimental evidence and different interpretations of experimental results. Boomarius et al suggested that from literature the major steps for the mechanism of cellulases are as follows;

1.Adsorption of cellulases onto the substrate through the binding domain (Stahlberg et al 1991).

2.Location of a bond susceptible to cleaving on the substrate surface (Jervis et al 1997). Chain end if cellobiohydrolase, cleavable bond if endoglucanase.

3.Formation of the enzyme-substrate complex ( By threading of the chain end into the catalytic tunnel (Divne et al 1998, Mulakala and Reilly 2005).

4.Hydrolysis of the beta glucosidic bond and simultaneous forward sliding of the enzyme along the cellulose chain (Divne et al 1998, Mulakala and Reilly 2005)

5.Desorption of cellulases from the substrate or repetition of step 4 or 2/3 if only the catalytic domain detaches from chain.

Figure 2.2(Reinikainen et al 1992) Steps 1 to 4 for a cellobiohydrolase acting on a cellulosic substrate (not drawn to scale). For endoglucanase, steps 2 and 3 are different as it does not require chain ends to act on.Step 1 — Adsorption, step 2 — location of chain end, step 3 — formation of enzyme–substrate complex, and step 4 — hydrolysis of the β-glycosidic bond. (Note: In step 3, some authors have suggested the possibility for the cellulose chain to thread into the catalytic domain by going over the binding domain.

Hydrolysis of cellobiose to glucose by β-glucosidase (if present in enzyme mixture). Product inhibition from cellobiose or glucose is also believed to affect the above mentioned steps. Changes in the properties of the substrate are also believed to affect hydrolysis too.

The role of the cellulose binding module in the initial stage of enzymatic hydrolysis has been highlighted in literature(Arantes and Saddler 2010). Rather than sequential shaving of cellulose fibrils as postulated by several studies, it has been suggested that the recalcitrance cellulose surface is first disrupted or loosened by non-hydrolytic proteins in this case cellulose binding module and this leads to an increase in cellulose surface area and making it more accessible to the cellulase enzyme complex in a mechanism termed amorphogenesis. The term amorphogenesis is coined by Coughan in 1985 to suggest a mechanism in which the dispersion, swelling or delamination of cellulose substrate occurs resulting in the reduction of the degree of fibrillar aggregation and/or crystallization and the creation of a larger surface by increasing the reactive internal surface. Fig 2.3A shows the inaccessible bulk of the cellulose being loosened up while they remain molecularly unchanged and then the cellulose network accessible to cellulase enzymes are then cleaved by the synergistic action of endo and exo- glucanases to soluble cello-olligisaccharides(fig 3b) which are further hydrolysed to cellobiose fig 3c and finally to glucose by beta glucosidase. The role of the cellulose binding module in the dispersion is proposed by some researchers(Klyosov and Rabinovich 1982, Rabinovich 1980). They proposed that cellulases are adsorbed to cellulose defects(disturbance in the crystalline structure of cellulose) and then the cellulose binding module penetrates into the interfibrillar space.

Figure 2.3 Schematic representation of amorphogenesis of cellulose fibers mediated by the carbohydrate-binding module (CBM) ofcellobiohydrolase I (CBHI) (adapted from Esteghalian et al 2000). For clarity, the carbohydrate-binding module is oversized compared with the catalytic domain

This causes a mechanical action due to the presence of large enzyme within the narrow space leading to swelling in the cellulose structure and allowing more water molexules between the fibrils. The water molecules penetrates further into the capillary space breaking the hydrogen bonds between the cellulose chain therefore increasing the surface area for the catalytic module to act upon the chain ends for cellobiohydrolase and random cleaving by endoglucanase.

Although a complete mechanism for this reaction has not been accepted, the three enzymes ;endoglucanase, cellobiohydrolase and beta glucosidase all work to produce soluble cellobiose through physical and chemical changes in the residual solid phase cellulose.

Chemical changes in cellulose are manifested as changes in degree of polymerisation and chain ends concentration. Endoglucanase increases the concentration of chain ends and significantly decreases the degree of polymerisation by attacking interior

portions of cellulose molecules while exoglucanases shorten degree of polymerisation incrementally and only occasionally decrease the concentration of chain ends. It can be concluded that endoglucanases is mostly responsible for chemical changes in cellulose over the course of the reaction but plays a smaller role in solubilisation however exoglucanase is believed to be primarily responsible for solubilisation but has a smaller effect in changing the chemical properties of residual cellulose(Zhang and Lynd 2004)

2.5.2 RATE LIMITATIONS AND SUBSTRATE PROPERTIES

Studies have shown that product inhibition by cellobiose, glucose and ethanol does not account for the significant rate drop associated with cellulose hydrolysis. Enzyme deactivation, decrease in accessible surface area, loss of synergy between cellulases and innacessibility caused by lignin and hemicelluloses all contribute to the rate slow down. It is also believed that cellulases adsorb unto lignin. (Boomarius 2009)

Kinetic models that account for adsorption have been proposed. The Langmuir isotherm offers the most common description of cellulase adsorption by a single adsorption equilibrium constant and a specific adsorption capacity.

Eb = Emax KadEfSc

1+ Kad Ef

in which Eb is adsorbed cellulase (mg or mol cellulase/L), Emax is the maximum cellulase adsorption per g cellulose (mg or mol cellulase / g cellulose), Sc iscellulose concentration (g cellulose/L), Ef is free cellulase (mg or mol cellulase/L), and Kad is the dissociation constant in terms of L/g cellulose.

Langmuir isotherm provides a good fit to data in most cases and can be used to compare the characteristic properties of various cellulose-cellulase system. It is however used only as a mathematical expression because it does not comply with its underlying assumptions; partially irreversible cellulase adsorption, interactions among adsorbing cellulase components especially at high concentration, multiple type of adsorption sites, entrapments of cellulase by cellulose pores and multiple component cellulase adsorption in which each component has a different constant(Zhang and Lynd 2004). Although several kinetic models like the 2 site adsorption model (Linder et al 1996, Medve et al 1997,Starhbeg et al 1991), freuldlich isotherm(Medve et al 1993), Langmuir freudlich isotherm(Medve et al 1993) have been proposed. None has accounted for the rate slowdown.

There are theories that suggest that cellulose can be classified as amorphous and crystalline. Crystallinity is the ratio of the amorphous cellulose to the crystalline cellulose. This theory assumes that amorphous cellulose is degraded faster than crystalline cellulose. Lynd et al 2002 reported that hydrolysis by fungal cellulases are typically three to thirty times faster than amorphous cellulase when compared to high crystalline cellulose and so crystallinity has often been thought of as providing an indication of substrate reactivity. However if this theory is correct then it should be expected that crystallinity should increase over the course of the reaction as a result of preferential reaction of amorphous cellulose(Betrabet and parkliker 1979, Oeshina et al 1983). Other studies have however found that crystallinity does not increase during enzymatic hydrolysis(Lenze et al 1990, Ohmine et al 1983, Puls and Wood 1991) and so it cannot be concluded that crystallinity is a key determinant of cellulose hydrolysis.(Lynd 2002).

Much of the research carried out on enzymatic hydrolysis and centred on rate limitation have been carried out on pure cellulose substrate and as such the effect of lignin and hemicelluloses have not been adequately taken into account. The method of analysis of substrate properties like crystallinity, accessible surface area and degree of polymerisation in lignocelluloses biomaterials are still unreliable. The concentration of accessible chain ends which can be a good indication of reactivity is not even known or taken into account in most of the models available in literature.

The diffusion rates of enzymes are believed to be affected by the presence of lignin and hemicelluloses. The substrate heterogeneity and the partial ‘’crystallinity’’ are all factors that contribute to this hydrolysis reaction occurring in one dimension (Figure 2.4 below). Reactions of this type are described by a phenomenon termed fractal kinetics.

Fig. 2.4. Schematic Michaelis, fractal, and ‘‘jammed’’ reactions. (A) Michaelis scheme. E, enzyme; S, substrate; ES, Michaelis intermediate; P, product. (B)Fractal scheme for an enzyme (ellipsoid) acting on chained substrates (dashed curve). The enzyme binds to the chain through its active site tunnel. After cleaving off a substrate unit (bar), the enzyme slides along the chain (in the direction) for the next catalytic cycle. (C) ‘‘Jammed’’ scheme for enzymes (ellipsoids) acting on substrate chains (dashed lines) that are packed orderly with defined spacing. Being ‘‘oversized’’ in comparison to the interchain space, the enzymes anchored on adjacent chains may jam each other (Xu and Ding 2006).

fractal kinetics is said to occur when reactions take place in constrained media giving rise to non uniform mixed reaction, apparent rate orders and time independent rate constants( Anaker and Kopelman1987, Kopelman 1986)

2.6.0 CELLULOSE AND STARCH HYDROLYSIS: A COMPARATIVE REVIEWStarch molecules are similar to cellulose molecules. There are glucose molecules linked by alpha-1,4 and alpha-1,6 glucosidic bonds unlike cellulose where the glucose molecules are linked by beta-1,4-glucosidic bonds (Fig 2.5). Starch molecules have different structures due to the two kinds of linkages; the alpha 1,4 and the alpha 1,6 glucosidic bonds. A straight chained polymer of glucose with only alpha 1,4 glucosidic bond is called amylose whereas a branched chained glucose polymer containing alpha -1,6 glucosidic linkages results in a branched polymer called amylopectin(fig 2.6). Branching in amylopectin occurs in approximately one per twenty five glucose units in the unbranched segments.

Fig2.5 (http://www.daviddarling.info/images/cellulose_starch.gif retrieved 2010-09-17)

Fig 2.6 structure of amylopectin(http://www.vivo.colostate.edu/hbooks/pathphys/digestion/basics/polysac.html retrieved 2010-09-17)

Starch is known to be insoluble in water at room temperature. This is because of the intermolecular and intramolecular hydrogen bonding present in the molecules and so penetration by water and other hydrolytic enzymes is not possible however when an aqueous suspension of starch is heated, the hydrogen bonds are weakened and water molecules are absorbed leading to swelling. A process commonly called gelatinization.Enzymatic hydrolysis of starch by alpha amylase enzymes can yield shorter chains of starch depending on the relative position of the bond under attack as counted from the chain end. The products vary from dextrin, maltotriose, maltose to glucose(wang )Unlike cellulase enzymes, alpha amylase enzymes are produced from a wide variety of organisms including human. They are therefore widely synthesized in nature and represent about 30% of the world’s enzyme production. (Dilek et al 2004).Several studies have reported that the rates of starch hydrolysis can be about 100 fold faster than the hydrolysis rate of cellulose. The hydrolysis rates of starch and cellulose can be compared on the basis of the following factors; the fraction of bonds accessible to the enzymes, the availability of chain ends and the solubilities of the hydrolysis products.The fraction of accessible glucose-glucose bonds as estimated by fujii et al 1981 for cellulose is believed to be 8-500 fold lower than for soluble starch. They also reported cellulose to be 5-200 fold less soluble than insoluble starch. This in turn affects the chain end availability ( per unit mass). This is because cellulose has a high degree of polymerization with a ratio of glucosyl units per chain ranging from 300-2000 however starch exhibits branching as noted earlier and each branch gives rise to a new chain end and the ratio of glucosyl units to chain ends is approximately 22 (branching for starch occurs every 17-26 glucose units). (Bertoldo and Antranikian 2002, Buelen et al 1998). The cellulose hydrolysis is

thus limited by the availability of chain ends for cellobiohydrolase (Zhang and Wilson 1997, Valjamae et al 2001, Schulein 2000). Cello-oligosaccharides(cellodextrins) are essentially insoluble at Degree of Polymerisation greater than 6-10 (Miller 1963, Pereira et al 1988, Zhang and Lynd 2003) however maltooligosaccharides are soluble at degree of polymerisation of 60 (John et al 1982). Again, the planar linear structure of the cellodextrins as compared to the helical branched structure of starch leads to a situation where many bond cleavages need to occur for soluble hydrolysis products to be generated. However for starch molecules, fewer bond cleavages have to take place before soluble products are generated. The enzymes also act on liquid starch rather than on solid in cellulose.The differences observed in hydrolysis rate between starch and cellulose do not lie in the intrinsic difference between the alpha linked glucosidic bonds and the beta linked glucosidic bonds but rather it is the difference in substrate characteristic in starch and cellulose that make the difference (Zhang and Lynd 2004).

2.7.0 ENZYMATIC HYDROLYSIS AND FERMENTATION OF CASSAVA PEELSThe composition of cassava peels have been reported as shown in tables 2.2-2.4 below

Table 2.2 Baah et al 1999

Constituents % Composition

Dry matter 86.5-94.1

Organic matter 91.1

Hemicellulose 29.0

Cellulose 20.8

Acid insoluble lignin 2.8

Acid soluble lignin 5.0

Others;Ca,K,Mg,Cu,Mn,N

2.0

Starch ?

Table 2.3 Aderemi and Nworgu 2007

Constituents % Composition

Dry matter 28.0

Crude Protein 5.4

Crude Fat 1.2

Crude Fibre 20.7

Ash 5.8

Reducing sugar 1.3

Carbohydrate(by difference)

66.9

Table 2.4 Ofuya and Obilor 1992

Note: ADF:acid detergent fibre, ADL:acid detergent lignin

The figures reported by these researchers show considerable differences. Although the composition of the peels might depend on the processing conditions of the tubers, it is expected that the differences would not be that much. These researchers failed to estimate the composition of starch in their analysis. We can only assume the starch composition by estimating the difference between organic matter and the lignin, cellulose and hemicelluloses which is inadequate. The studies would be more useful if the exact composition of starch, cellulose and hemicelluloses is known. A further distinction in the breakdown of the sugars in the hemicellulose component would also give an idea of the complete biomass characterization of cassava peels. This would then

Constituents % Composition

Cellulose 5.40 + 1.34

Hemicellulose 21.65 +3.15

ADL (Lignin) 4.81 + 0.28

ADF 9.76 + 1.21

Crude Protein 5.35 +1.21

Starch ?

give an idea of the ratio and amount of enzymes needed in an enzyme cocktail for the hydrolysis reaction. The analysis by Baah et al 1999 in Table 2.2 represents the most realistic result in literature.The composition shows that cassava peels can be characterised as a complex substrate with starch, cellulose, hemicelluloses, lignin and pectins all present in varying proportions each with its own unique chemical bonds of different strengths. It has been suggested in several studies of the imperative need to combine a cocktail of enzymes to achieve a higher saccharification yield for lignocelluloses. The studies reported so far in literature on the enzymatic hydrolysis of cassava peels (Yoonan and Kongkiattikajorn 2004, Sornvoraweat and kongkiattikajorn 2009) show that a combination of enzymes(alpha amylases, cellulase, pectinase and xylanase) were employed for saccharification. The researchers analysed the reducing sugars produced however these studies do not provide sufficient information on the interaction mechanism of the various enzymes either amongs themselves or with the amorphous and crystalline parts of the peel. The studies might have been far more useful if they included the HPLC analysis of the released sugars. This would have provided a more detailed understanding of the saccharification process. A study of the concentration of the chain ends and the sugar release patterns of the enzymes is necessary to fully comprehend the mechanism of this reaction.A closer look at the study by Sornvoraweat and Kongkiattikajorn 2009 reveals that the research centred on the simultaneous saccharification and fermentation process rather than on optimizing the sugar yield from the saccharification step. An enzyme mixture of cellulase, xylanase and pectinase were used for the hydrolysis on pretreated cassava peels. The addition of alpha amylase would have improved the reducing sugar yield from the maximum yield of 72% reported. This maximum yield was reported for sulphuric acid pretreated cassava peels. Alkaline pretreated and hot water pretreated peels yielded % and_% respectively. The simultaneous saccharification and fermentation was carried out at 300C and a pH of 5.5. Perhaps, the study would have also been carried out at different temperatures and pH to determine the optimum temperature and pH for the enzymes and the microorganism, saccharomyces cerevisiae.The study by Yoonan and kongkiattijorn 2004 compared the enzymatic hydrolysis by alpha amylase and glucosidase on one hand and the hydrolysis of cellulase and pectinase on the other hand. The former yielded 75.41% of reducing sugar and the latter yielded 43.39% reducing sugars. Both sets of enzymes were not combined. The use of acid and alkaline for hydrolysis was also experimented. Although it is agreed by the researchers that hydrolysis by acid and alkaline was not as complex as enzyme hydrolysis especially when a cocktail

of enzymes with different optimum operating conditions are required, the maximum reducing sugar production was lower with acid or alkaline hydrolysis. Moreover, the severe experimental conditions that require equipments to withstand corrosion makes the process expensive.As highlighted earlier in section 2.3, the need to relate the effects of enzymatic hydrolysis on substrate properties would also be of immense help to understanding the kinetics of this unique substrate.

2.8.0 MICROBIAL FERMENTATION BY ZYMOMONAS MOBILISEthanol fermentation by yeast is an art known since the stone age and it involves the process where chemical changes are brought about in an organic substrate in this case carbohydrate through the action of enzymes secreted by yeast.Saccharomyces cerevisiae and candida tropicalis are the only microorganisms of fermentation reported to be used on cassava peels to digest glucose and xylose.In recent years, there has been a growing interest in the use of zymomonas mobilis, a gram negative bacteria. Zymomonas mobilis can be isolated from the African palm wine, spoilt beer in European countries and from alcoholic drinks; tequila and pulque.A comprehensive review by Gunasekaran and Chandra-raj 2003 shows that zymomonas mobilis has the following advantages over a large variety of yeast; It has a higher sugar uptake and ethanol yield, it also has a higher ethanol tolerance and does not require the controlled addition of oxygen during fermentation.Another major advantage Z mobilis is believed to have is its amenability to genetic manipulation. D-xylose genes from xanthomonas was introduced into z.mobilis CP4 with other pentose phosphate pathway enzymes like xylose isomerise,xylulokinase,transketolase and transaldolase. This engineered strain yielded 0.44g ethanol/g xylose corresponding to 86% of the theoretical yield. Recombinant DNA technology is still being exploited currently to produce ethanol from starch, cellulose,lactose,mannose and other hemicelluloses sugars with varying amount of success. (Zhang et al 1995).The use of zymomonas mobilis on cassava peels is not reported although defranca et al 1996 investigated different strains of z.mobilis on cassava flour and found that the strain CP3 was the best with an ethanol yield of 0.48g/g and productivity of 4.14g/l/h.The tolerance of zymomonas mobilis to ethanol at high concentarion might also indicate that the microorganism will be a good candidate for cellulosic ethanol fermentation. A large variety of yeast have been reported to be inhibited by

byproducts from pretreatment methods like steam explosion or acid hydrolysis. Zymomonas mobilis might be a better alternative fermentative organism.This study will compare the fermentation abilities of zymomonas mobilis and saccharomyces cerevisiae although integrating the pretreatment, hydrolysis and fermentation steps to streamline the entire process and reduce cost still remains a challenge.

CHAPTER THREE

3.0.0 AIMS AND OBJECTIVES

This research will aim at identifying and optimizing the appropriate pretreatment method for cassava peels based on its physico-chemical composition. The research will seek an understanding of the effects of pretreatment and enzymatic hydrolysis on ethanol production from cassava peels.

This project will also aim at seeking a better understanding of the interactions between the starch, cellulose, hemicellulose and lignin as the current understanding of the interaction between the crystalline and non crystalline part of the cassava peel is still not clear.

Our aim would be to develop a strategy for enzymatic degradation of the peels by the combination of a suitable enzyme cocktail of amylases, cellulases, hemicellulases, pectinases as well as trying out the effect of other enzymes like lacasses, ligninases and peroxidises on hydrolysis yield and rate. We would also explore the use of surfactants to see its effect on limiting the decreasing yields of hydrolysis. We would also seek a better understanding of the decreasing rates usually observed with cellulose hydrolysis with HPLC analysis of the released sugars and hope for some understanding relevant more widely to lignocellulose hydrolysis.

Enzymatic assays will be studied to gain an understanding of enzyme and substrate variables like substrate concentration, enzyme concentration, degree of synergism amongst others with a view of identifying rate limiting factors.

Finally, we will seek to integrate the hydrolysis reaction with the fermentation reaction to streamline the entire process using simultaneous saccharification and fermentation (SSF) with Zymomonas mobilis.

REFERENCES

Adegbola, A.A.,and V.O. Asaolu. 1986. Preparation of cassava peels for use in small ruminant production in Western Nigeria. In: Toward Optimum Feeding of Agricultural By-products to Livestock in Africa. Preston, T.R., Nuwanyakpa, M.Y.(Eds.), pp. 109-115.Proc. of Workshop held at the University of Alexandria, Egypt. Oct. 1985. ILCA, Addis Ababa, Ethiopia.

Al-Zuhair S. The effect of crystallinity of cellulose on the rate of reducing sugars production by heterogeneous enzymatic hydrolysis. Bioresource Technol 2008;99: 4078–85.

Anacker LW, Kopelman R. Steady-state chemical kinetics on fractals: segregation of reactants. Phys Rev Lett 1987;58:289–91.

Bader J, Bellgardt KH, Singh A, Kumar PKR, Schugerl K. Modelling and simulation of cellulase adsorption and recycling during enzymatic hydrolysis of cellulosic materials. Bioprocess Eng 1992;7:235–40.

Beldman G, Voragen AGJ, Rombouts FM, MFS-v Leeuwen, Pilnik W. Adsorption and kinetic behaviour of purified endoglucanases and exoglucanases from Trichoderma viride. Biotechnol Bioeng 1987;30:251–7.

Bommarius AS, Katona A, Cheben SE, Patel AS, Ragauskas AJ, Knudson K, et al. Cellulase kinetics as a function of cellulose pretreatment. Metab Eng 2008;10:370–81.

Brown RF, Holtzapple MT. A comparison of the Michaelis–Menten and HCH-1 models. Biotechnol Bioeng 1990;36:1151–4.

Divne C, Ståhlberg J, Teeri TT, Jones TA. High-resolution crystal structures reveal how a cellulose chain is bound in the 50 Å long tunnel of cellobiohydrolase I from Trichoderma reesei. J Mol Biol 1998;275:309–25.

Duff, S.J.B., Murray, W.D., 1996. Bioconversion of forest products industry waste cellulosics to fuel ethanol: a review. Bioresour. Technol. 55, 1–33.

Fan, L.T., Gharpuray, M.M., Lee, Y.-H., 1987. In: Cellulose Hydrolysis Biotechnology Monographs. Springer, Berlin, p. 57.

Galbe, M., Zacchi, G., 2007. Pretreatment of lignocellulosic materials for efficient bioethanol production. Adv. Biochem. Eng. Biotechnol. 108, 41–65.

Ghose TK, Bisaria VS. Studies on the mechanism of enzymatic hydrolysis of cellulosic substances. Biotechnol Bioeng 1979;21:131–46.

Gunasakaran P and chandraraj K 1999. Ethanol fermentation technology-zymomonas mobilis current science 77, 56-58

Holtzapple, M.T., Davison, R.R., Stuart, E.D., 1992b. Biomass refining process, US patent 5, 171, 592.

Hong J, Ye X, Zhang YHP. Quantitative determination of cellulose accessibility to cellulase based on adsorption of a nonhydrolytic fusion protein containing CBM and GFP with its applications. Langmuir 2007;23:12535–40.

Hong J, Ye X, Zhang YHP. Quantitative determination of cellulose accessibility to cellulase based on adsorption of a nonhydrolytic fusion protein containing CBM and GFP with its applications. Langmuir 2007;23:12535–40.

Jervis EJ, Haynes CA, Kilburn DG. Surface diffusion of cellulases and their isolated binding domains on cellulose. J Biol Chem 1997;272:24016–23.

Jervis EJ, Haynes CA, Kilburn DG. Surface diffusion of cellulases and their isolated binding domains on cellulose. J Biol Chem 1997;272:24016–23.

Kadam KL, Rydholm EC, McMillan JD. Development and validation of a kinetic model for enzymatic saccharification of lignocellulosic biomass. Biotechnol Progr 2004;20:698–705.

Kopelman R. Fractal reaction kinetics. Science 1988;241:1620–6.

Kopelman R. Rate processes on fractals: theory, simulations and experiments. J Stat Phys 1986;42:185–200.

Kristensen, J.B., Borjesson, J., Bruun, M.H., Tjerneld, F., Jorgensen, H., 2007. Use of surface active additives in enzymatic hydrolysis of wheat straw lignocellulose. Enzyme and Microbial Technology 40, 888–895.

Laidler KJ. Theory of the transient phase in kinetics, with special reference to enzyme systems. Can J Chemistry 1955;33:1614–24.

Luo J, Xia L, Lin J, Cen P. Kinetics of simultaneous saccharification and lactic acid fermentation processes. Biotechnol Progr 1997;13:762–7.

Lynd LR,Weimer PJ, WHv Zyl, Pretorius IS. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol R 2002;66:506–77

Mackie, K.L., Brownell, H.H., West, K.L., Saddler, J.N., 1985. Effect of sulphur dioxide and sulphuric acid on steam explosion of aspenwood. J. Wood Chem. Technol. 5, 405–425.

Mansfield SD, Mooney C, Saddler JN. Substrate and enzyme characteristics that limit cellulose hydrolysis. Biotechnol Progr 1999;15:804–16.

McMillan, J.D., 1994. Pretreatment of lignocellulosic biomass. In: Himmel, M.E., Baker, J.O., Overend, R.P. (Eds.), Enzymatic Conversion of Biomass for Fuels Production. American Chemical Society, Washington, DC, pp. 292–324.

Mes-Hartree, M., Dale, B.E., Craig, W.K., 1988. Comparison of steam and ammonia pretreatment for enzymatic hydrolysis of cellulose. Appl. Microbiol. Biotechnol. 29, 462–468

Millet, M.A., Baker, A.J., Scatter, L.D., 1976. Physical and chemical pretreatment for enhancing cellulose saccharification. Biotech. Bioeng. Symp. 6, 125–153.

Morjanoff, P.J., Gray, P.P., 1987. Optimization of steam explosion as method for increasing susceptibility of sugarcane bagasse to enzymatic saccharification. Biotechnol. Bioeng. 29, 733–741.

Mosier, N., Hendrickson, R., Ho, N., Sedlak, M., Ladisch, M.R., 2005a. Optimization of pH controlled liquid hot water pretreatment of corn stover. Bioresour. Technol. 96, 1986–1993.

Mosier, N., Wyman, C.E., Dale, B.D., Elander, R.T., Lee, Y.Y., Holtzapple, M., Ladisch, C.M., 2005b. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour. Technol. 96, 673–686.

Mulakala C, Reilly PJ. Hypocrea jecorina (Trichoderma reesei) Cel7A as a molecular machine: a docking study. Proteins Struct Funct Bioinf 2005;60:598–605.

Nartey,F., 1979. Studies on cassava cynogenesis, and biosynthesis of cynogenic glucoside in cassava (Manihot spp.), In: Nestel, B., Maclntyre, R. (Eds.), Chronic cassava toxicity, IDRC 010e: 73-87.

Prescott S. C and Dunn C.G (1940) Industrial Microbiology First Ed – McGraw Hill Book Company Pp 1 – 10

Saha, B.C., Iten, L.B., Cotta, M.A., Wu, Y.V., 2005. Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol. Process Biochem. 40, 3693–3700.

Sánchez, Ó.J., Cardona, C.A., 2008. Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresour. Technol. 99, 5270–5295.

Schnell S. A century of enzyme kinetics: reliability of the KM and vmax estimates. Comment Theor Biol 2003;8:169–87.

Ståhlberg J, Johannson G, Pettersson G. A new model for enzymatic hydrolysis ofcellulose based on the two-domain structure of cellobiohydrolase I. Nat Biotechnol 1991;9:286–90

Sun Y and Cheng J. 2002, Hydrolysis of lignocellulosic materials for ethanol production; a review. Bioresource technology (83) 1-11

Tu M, Chandra RP, Saddler JN. Evaluating the distribution of cellulases and the recycling of free cellulases during the hydrolysis of lignocellulosic substrates. Biotechnol Progr 2007;23:398–406.

Väljamäe P, Sild V, Pettersson G, Johansson G. The initial kinetics of hydrolysis by cellobiohydrolases I and II is consistent with a cellulose surface — erosion model. Eur J Biochem 1998;253:469–75.

Van Walsum, P., Allen, S., Spencer, M., Laser, M., Antal, M., and Lynd,L. 1996. Conversion of lignocellulosic pretreated with liquid hot water to ethanol. Appl. Biochem. Biotechnol. 57/58:157-170.

Wald S, Wilke CR, Blanch HW. Kinetics of the enzymatic hydrolysis of cellulose. Biotechnol Bioeng 1984;26:221–30.

Wooley, R., Ruth, M., Glassner, D., Sheehan, J., 1999. Process design and costing of bioethanol technology: a tool for determining the status and direction of research and development. Biotechnol. Prog. 15, 794–803.

Wyman, C.E., 1996. Handbook on Bioethanol: Production and Utilization. Taylor Francis, Washington, p. 417.

Yang, B., Wyman, C.E., 2008. Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels Bioprod. Bior. 2, 26–40.

Yang, B., Wyman, C.E., 2008. Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels Bioprod. Bior. 2, 26–40.

Yang, B., Wyman, C.E., 2008. Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels Bioprod. Bior. 2, 26–40.

Zhang YHP, Lynd LR. A functionally based model for hydrolysis of cellulose by fungal cellulase. Biotechnol Bioeng 2006

Zheng, Y.Z., Lin, H.M., Tsao, G.T., 1998. Pretreatment for cellulose hydrolysis by carbon dioxide explosion. Biotechnol. Prog. 14, 890–896.